CN1092866A - A kind of based on light scattering and there is not the immune analysis method of particle self aggregation situation - Google Patents
A kind of based on light scattering and there is not the immune analysis method of particle self aggregation situation Download PDFInfo
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- CN1092866A CN1092866A CN93119931A CN93119931A CN1092866A CN 1092866 A CN1092866 A CN 1092866A CN 93119931 A CN93119931 A CN 93119931A CN 93119931 A CN93119931 A CN 93119931A CN 1092866 A CN1092866 A CN 1092866A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/553—Metal or metal coated
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/02—Investigating particle size or size distribution
- G01N15/0205—Investigating particle size or size distribution by optical means
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Abstract
A kind ofly measure one or more antibody in the fluid sample simultaneously, the homogeneous immune analysis method of antigen or haptens analyte comprises the influence that the above-mentioned analyte of quantitative test changes light scattering pulse height distribution histogram statistical.This histogram change be by larger-diameter monodispersity binding molecule bag by the poly microsphere by than the polydispersity binding molecule bag of minor diameter by colloidal metal particulate institute combination generation afterwards.For analyzing multiple analyte simultaneously, distribute the microsphere of different-diameter or refractive index for every kind of analyte, this analytical approach can be used in the system of forward combination, replacement, inhibition, type of competition, is changed to instruct by the histogram dimension that depends on system and finishes.
Description
The present invention is chiefly directed to the homogeneous immune analysis method of measuring a kind of antigen, antibody or haptens analyte in the fluid sample, is to realize by measuring the light scattering signal that the monodispersity particulate sends in the particle analysis gauge that flows.Especially special is the variation that this invention relates to the measuring light scattered signal, this light scattering signal is from the monodispersity poly microsphere of binding molecule bag quilt, and this is these microspheres and the middle result who combines of analyte of the polydispersity colloidal metal particulate of binding molecule bag quilt.
Antibody, antigen and many haptens present high affinity, and this is not only to their complementary albumen, and to some solid state surface, for example the surface of the aperture of plastics microtitration flat board, plastic test tube wall, poly microsphere and colloidal metal particulate also is like this.A revolution in diagnostic analysis method field has been drawn in the exploitation of these characteristics, and these analytical approachs are used for analyzing the analyte that resembles the such fluid sample of serum recited above.
On solid-state holder, carry out the ability the earth to the utmost of antigen antibody interaction and simplified the step of from untapped reactant and interfering material, isolating the immunocomplex that comprises analyte.Normal some chaff interferences like this that exist in the biologicfluid sample.These systems are commonly referred to as " solid-phase immunoassay " or " immunosorbent analysis ", belong to " heterogeneous immunoassay ".Phase separation step in the heterogeneous immunoassay is very valuable aspect the interference that reduces the non-specific binding material, these materials have adverse effect to the sensitivity of analytical approach usually, these analyze trouble and expensive, and are the focuses of integrity problem in the automated system.
In addition, these heterogeneous systems also have a shortcoming, promptly require the part of immunocomplex or another part to be easy to quantitative molecular labeling by a kind of.These molecules are commonly referred to as " reporter molecules ", comprise radioactive isotope (radiommunoassay, RIA), and enzyme (common enzyme-linked immunoassay with chromophore, ELISA), fluorescence molecule (fluoroimmunoassay, FIA), chemiluminescent molecule (CIA), gold grain, photosensitive molecular or the like.As review, can be with reference to Kemeny, D.M., et al., Immunology Today, 7:67(1986).Further, because limited as the number of the chromophore of acceptor molecule and fluorophore, and the emission spectrum of these molecules is overlapping in a large number, so analyze when being unsuitable for carrying out multiple analyte with these reporter molecules.For example, Cambridge Biotech. to C.difficile Toxin A and Toxin B, HTLV-I and HTLV-II, it is exactly inseparable that simultaneity EIA that HIV-1 and HIV-2 carry out analyzes.The present invention has simplified from single reaction mixture the separability of signal in the multiple analyte widely.
The immunoassay that " homogeneous immunoassay " this term is used for not being separated.These systems comprise the microparticle agglutination analysis in conjunction with albumen bag quilt, because they need less step when realizing robotization, and robotization is all simple aspect machinery, flowability, electronics, so of great use.The example of immunoassay that need not phase separation step is as follows: the aggegation of latex microsphere body, hemagglutination and the depolarized analysis of fluorescence.The example of analyzing for single latex beads aggegation of planting analyte can be 4521521 in the patent No., 4184849,4279617, find on 4191739 and 4851329 the United States Patent (USP), can on the U.S. Patent application (application number is 883574) of the issued for approval of Hansen, find for the example of the analysis of multiple analyte in the single liquid sample.Be used for the nephelometric turbidity of aggegation or the depolarized analysis of fluorescence or the turbidimetric assay automatic system is simple and expenditure of construction is cheap, different with heterogeneous analysis is that it does not need to maintain continually complicated phase-separating device.
Yet, say from another point of view, because the existence of interfering material in the body fluid, this method of homogeneous immunoassay can not be satisfied the high sensitivity requirement that many important medical science detect, as not satisfying the requirement of enzyme-linked immuno assay (ELISA) and radioimmunoassay (RIA).Can look at for example Masson et al. to the review of this problem, Methods in Enzvmology, 74:115(1981) with Collett-Casssart et al., Clin Chem., 27:64(1981).An importance of the present invention is that it is a kind of material immune analysis method that is not subjected to non-specific interference, and sensitivity can reach 5 * 10 at least
-13M.This level of sensitivity is than the original high 2-3 of a homogeneous latex beads aggegation analytical technology order of magnitude.Look at several examples: α-fetoprotein (3 * 10
-10M, above-mentioned Collett-Cassart etc.), human chorionic gonadotrophin (HCG) (6 * 10 in the urine
-11M, Lentrichia et al, .J.Immunol.Meth., 89:657(1986) (but analyte level two decuple when marking sensitivity limit, only show 87% correlativity with radiommunoassay (RIA)), the serum digoxin (digoxin) (3 * 10 that the depolarized method of fluorescence is measured
-10M, S.Wong in D.Chan, ed., Immunoassay Automation, Academic Press, 1992, P.329).
In the past generally can not be satisfactory for eliminating or reduce the technical method that non-specific chaff interference does the adverse effect of homogeneous immunoassay.These methods comprise: the high magnification dilution of humoral sample (Fritz et al., J.Immunol., 108:110(1972)), but can reduce to equal proportion sensitivity like this; The use antibody fragment (Masson, Id.), but this method is expensive and unreliable; Use conditions such as special P H, ionic strength and buffer type, (or) adding chelate or other cleanser (Masson, Id.), but can introduce the multiple dependence factor like this, all must be chosen to best to these factors of each analyte, this just makes that analytic process is extremely expensive and bothers (Lim et al .J.Clin.Chem.Clin.Biochem., 20:141(1982)).
Solve this difficult problem of non-specific interference and also have other method, come nonspecific reaction in the inhibition analysis process comprising the latex ultra micro spheroid that uses IgG bag quilt, this analytic process has been to use the latex spheroid aggegation of antibody fragment to analyze.It was reported, a kind ofly like this used the sensitivity of the aggegation method of Coulter principle resistance flow particles analyser to be about 5 * 10 with 30 μ m apertures
-13To 4 * 10
-12M(Sakai et al., Chem.Pharm.Bull., 37:3010(1989)).The weakness of this method is that addition reaction thing (nonspecific, IgG wraps the ultra micro spheroid of quilt) can constantly increase the expense of manufacturing, quality supervision and storage.The present invention can eliminate this critical weakness, it by in all a kind of reactants the specific immune response effect with improve specific effect and combine and realize.In addition, though Ku Shi (Coulter Principle) batch particle-counting system that Sakai etc. use produces quantitative result, when detecting aggegation, need use aperture (30 μ m), this has just produced a well-known problem, latch up phenomenon when being agglutinating reaction (Masson, Id.).The mobile particle analysis gauge of the sheath tubular type that the present invention uses has the aperture of 250-300 μ m, has eliminated latch up phenomenon.So, in people's such as Sakai method, if attempt to analyze simultaneously more than one analyte, then will to produce a problem-Ku Shi (Coulter) volume overlapping in the distribution of specific agglutination particulate (dimer, tripolymer etc.), and there is not this problem in the present invention, because polymer can not form, and can carry out multiple simultaneity analysis and do not need to calculate for eliminating the overlap problem student movement of counting.
Another problem that former aggegation immunoassay runs into is, during containing the reaction mixture complete reaction of a micron or above particulate, need stir that (masson, Id.), this just requires the harsh washing between the sample to hide mutually to prevent sample with mechanical stirrer.The further advantage of the present invention is at present with regard to the robotization aspect, finishes agglutinating reaction and do not need to carry out the stirring of sample in effectual time.
People's such as Schutt European patent 0254430 and United States Patent (USP) 5017009 have been put down in writing scattering total internal reflection (STIR) analytical approach to a kind of analyte, use the aurosol particle to come mark to be combined in protein on the visible plastic board that optical characteristics is arranged of coating in this method.This immunoassay relies on is that this ripple is caused by the aurosol label that is brought to the interphase place in immune response to a kind of measurement of light of being returned by the disturbance institute scattering of transient wave.This instantaneous result who is considered to the incident light wave total internal reflection, the shortcoming of this measuring method are that expensive optical characteristics plastic board can not be reused, and use once and just must abandon.
Also has an important requirement for the antigen in fluid sample, antibody, haptenic immune analysis method, promptly to combine the low cost of machinery simplification and the analysis of particle agglutination homogenieity, to reduce those chaff interference adverse effects that often exist in the heterogeneous analysis based on solid-state holder simultaneously, and want and than original aggegation analytical approach higher efficient and wideer measurement range to be arranged.This needs will be satisfied by the present invention who is described in more detail below.
The present invention relates to a kind of new immune analysis method, can detect and quantize the concentration of one or more antigens in the single fluid sample, haptens, antibody analyte simultaneously.The present invention is based on once accidental and unexpected observations, promptly when the molecule bag with respect to little polydispersity combined state is attached to big relatively monodispersity combined state by glue state metal particle molecule bag by poly microsphere surface on the time, the linearity of the light scattering pulse height distribution histogram of these poly microspheres changes, and these changes on the histogram linearity can be associated with the concentration that causes the analyte that the linearity changes.Light scattering preferably is low-angle forward light scattering basically or is the light scattering at right angle basically.For reaching this purpose suitable histogram dimension to be determined is the illustrated standardized peak width of histogram, promptly in the peak width at half place of peak height.
In a forward association reaction (interlayer reaction) embodiment, by the big poly microsphere of the monodispersity of the first binding molecule bag quilt, by the little glue state metal particle of polydispersity of the second binding molecule bag quilt, and and all complementary analyte of these two kinds of binding molecules between, just formed a monodispersity immunocomplex.Can the light scattering pulse height distribution histogram dimension of monodispersity particulate immunocomplex and as the histogram dimension of reference compare (its be under the situation that does not have metallic particles to exist by the monodispersity bag by the poly microsphere and before and after analysis is carried out or the connection analyte that detects in carrying out obtaining), the histogram dimension statistical that the existence because of analyte can also be caused increase and fluid sample in the concentration associated of this analyte.In an alternate embodiment of the present invention, by the monodispersity poly microsphere of analyte bag quilt with between by a kind of polydispersity colloidal metal particulate of anti-analyte antibody sandwich, a kind of immune complex reaction thing at first forms, measure the relative analyte of immunocomplex and expose its light scattering histogram dimension of front and back, the histogram dimension statistical change after its relative analyte exposes just can be closed with analyte concentration in the fluid sample and be linked up.In a competitive embodiment of the present invention, for being attached on the monodispersity poly microsphere, its by with the first binding molecule bag quilt of anti-analyte complementary antibody, analyte with competed mutually by the polydispersity colloidal metal particulate of anti-analyte quilt that antibody wraps.Reduce analyte if between microsphere and metal particle, form the immunocomplex stage, will reduce light scattering pulse height distribution histogram dimension pro rata with analyte concentration in the fluid sample.In an inhibitory reaction embodiment, this immunoassay depends on the inhibition ability of analyte, promptly suppresses to be attached on the monodispersity poly microsphere by binding molecule bag quilt by the polydispersity metal particle of anti-analyte antibody sandwich.
The present invention's is the forward combination that discloses above-mentioned creative method, details and the scope that substitutes, competes, suppresses embodiment on the one hand.
On the other hand, disclose poly microsphere and the colloidal metal particulate that is applicable to realization the inventive method.
Again on the one hand, disclose the example that adopts specific analyte in this creative method detection of biological liquid.
In still another aspect of the invention, disclosure is that this creativeness method is used to the multiple analyte of analyzing simultaneously in the same fluid sample in the single analyses process.
To also have on the one hand, to use the purpose of being not only in order analyzing when the metal particle that wraps quilt is promptly arranged, it can also serve as cleanser, and the non-specific interfering material that will be present in the biological sample is removed from the immunoassay reaction mixture expediently.
With reference to the detailed description of following most preferred embodiment and appended claim, more than and other purpose will be clearly.
Fig. 1 has represented poly microsphere and metal particle (A), and metal particle is attached to, and the light scattering pulse height of (B) and back (C) distributes before the monodispersity poly particulate.
Fig. 2 represents standardization peak width parameter and calibration (for example standard) curve that forward is analyzed in conjunction with (for example sandwich construction).
Fig. 3 represent typical curve (A) that a people's serum first shape arteries and veins promotes that hormone (TSH) is analyzed and with the comparison of other method.
Fig. 4 represent in the human serum a T4 typical curve (A) and with the comparison of other method.
Fig. 5 represents latex-latex agglutination analysis of an IgG.
Fig. 6 has represented the dynamics situation that aurosol particulate that analyte causes combines with the latex microsphere body.
The present invention is based on a kind of new immune analysis method of the particulate analysis method that flows, and is used for the analysis of fluid sample antigen, antibody and haptens analyte.This method has been utilized a chance and unexpected the discovery, promptly induce at analyte when relatively little polydispersity metal particle, with the proportional immuno-chemical reaction of its concentration in when being attached on the relative big monodispersity poly microsphere, statistical can take place and change in the measured value of some physical property of this poly microspheres preparation.The monodispersed particulate nature of can carrying out that is measured by the particle analysis gauge that flows is exemplified below: the so-called Ku Shi volume (Coulter-volume) of insulation particulate in the mobile particle analysis gauge of the resistance-type under a given hole diameter; Fluorescent emission when single particulate is shone by certain wavelength light in an optics flows particle analysis gauge; Monodispersity microsphere in an optics flows particle analysis gauge is substantially at scattering pulse height profile histogram of illuminated time of an assigned direction.Last character has been used on the new method of the present invention, and this new method is used for detecting simultaneously one or more antigens, antibody, the haptens analyte in the single fluid sample.This said last character also has been used in a kind of method of the harmful disturbing effect that can avoid or reduce non-specific material in the biological fluid, and these interference are present in the old aggegation immune analysis method.
The mobile particle analysis gauge (FPA) of the optics of Shi Yonging adopts the light scattering of sheath tubular type fluid hose and incident beam to come sensing and measures the formation degree that immunochemistry is induced complex in the present invention, this species complex forms between relatively little polydispersity colloidal state colloidal metal particulate and a large amount of big relatively monodispersity poly microsphere, the narrow sample flow that is directed flows through from the stream pipe, and incident beam is the scattered light of laser preferably.Preferably be low-angle forward light scattering or substantially substantially for the right angle light scattering.Measuring method is based on unexpected a discovery, promptly be attached to poly microsphere surface and when forming immunocomplex, these metal particles cause the very big change of the light scattering pulse height distribution histogram dimension of this monodispersity poly microsphere when above-mentioned polydispersity metal particle.And the polydispersity metal particle is under measuring condition of the present invention, the poly particle areas not scattered light (even at metal particle since with serum in after aggegation takes place in ever-present non-specific binding substance reaction).When the poly microsphere by first kind of complementary binding molecule bag quilt, metal particle by the another kind bag by the time, a kind of protein (for example antigen or antibody) or the haptens analyte all complementary with these two kinds of binding molecules will be linked to metal particle on the poly particulate, thereby cause the variation of the light scattering pulse height distribution histogram dimension of monodispersity poly microsphere, what compare with it is the reference histogram dimension that obtains under no metal particle and analyte situation.It is directly related with the quantity of the analyte that exists in the fluid sample to have been found that above-mentioned histogram dimension changes.
Above-mentioned variation takes place under the situation that the histogram dimension does not have obviously to move in the position of the light scattering pulse height distribution histogram of this monodispersity poly microsphere, and this poly collection situation that shows the poly microsphere is not tangled into, and this is an important discovery.
The best dimension that the monitoring histogram changes is the peak width at half the peak height place in the diagrammatic representation of light scattering pulse height distribution histogram.This dimension promptly is called " standardization peak width " or " NPW " (please see Figure the 2A example) in this manual.In above-mentioned forward combination (sandwich construction) immunocomplex formation method, the variation of histogram dimension is that the related coefficient of the deviation (CV) that centers on histogram mean value of widening and causing thus of NPW in its diagrammatic representation increases.
Also have been found that measurement that the statistical of light scattering pulse height distribution histogram dimension changes can be used as substitute, the basis of a kind of analyte in competition and the inhibition type immune analysis method in the quantitative measurement fluid sample.
In a substituted type embodiment,, prepare the immune complex reaction thing in advance by being attached to by the monodispersity poly microsphere of analyte bag quilt by the polydispersity colloidal metal particulate immunity of anti-analyte bag quilt.The baseline determination of the light scattering pulse height distribution histogram of this reactant is similar to the result who obtains when two based fine particles immunity compound tense with the dimension of expressing, that is to say, if detect be dimension it be a wide NPW.When this reactant with contain when being mixed mutually by the solution of the analyte of the binding molecule complementation of metal particle with bag, metal particle will be directly proportional with contained analyte concentration replacedly from the immune complex reaction thing get off.The histogram dimension will be got back to the sort of situation that is similar to according to monodispersity poly microsphere, and promptly the histogram dimension reduces, and that is to say NPW(and CV) will be reduced.Statistical degree that the dimension that a plurality of standard meanses can reflect NPW reduces and the analyte concentration associated in the fluid sample.
In a state of conflict embodiment, for the polydispersity metal particle that is attached to binding molecule bag quilt gets on, the monodispersity poly particulate and the analyte of binding molecule bag quilt are competed mutually.The NPW of poly particulate and the concentration of analyzed fluid sample are inversely proportional to, and that is to say, high analyte concentration will " be caught " metal particle of major part, only stay few relatively part metals particulate and combine with the poly microsphere.This can produce narrow relatively histogram, just a little NPW.In above two embodiment, analyte can be antigen, haptens or antibody, if instruct clue selection suitable binding molecule wraps by microsphere and metal particle according to provided by the invention, is the known technology of assay those skilled in the art.
In an inhibition type embodiment, will comprise the sample of analyte and the polydispersity metal particle of anti-analyte antibody sandwich and hatch certain hour, need not be with reaching required so much time of balance, may be on several hours the order of magnitude.Right using adds by the monodispersity poly microsphere of analyte bag quilt, continues to hatch another period, and the order of magnitude is a few minutes, is attached to the poly microsphere at this following period of time analyte inhibition metal particle and gets on.Under above situation, measure the histogram dimension.Because the analyte isolating metal particulate of high concentration, the result is that it has produced narrow histogram peak width; The analyte of low concentration then produces adverse consequences.
As mentioned above, the important feature of the embodiment of the invention is that used mixtures incubated does not during reaction need to stir.When adding the reacted constituent of reaction mixture, only need simple agitation (1-2 minute) so that potpourri is even.
Multiple commercial poly particulate can use in the present invention, but the best is homogeneous latex microsphere body.Bangs, L.B., homogeneous present latex particulate, Seragen, Indianapolis, 1984.Though " latex " this term strictness is meant the polyisoprene of forming emulsion in fact, its implication has been expanded, and comprises the synthetic polymer such as polybutadiene, polystyrene here.The mean diameter of homogeneous latex microsphere body is preferably in 0.5-5.0 μ m between 0.05-10 μ m, and has stable hydrophobic surface group, combines protein above tightly, thereby produces stable water wettability colloidal state suspending liquid.More than be the suitable diameter that uses among the present invention, though commercial latex microsphere body diameter can reach 100-120 μ m.Latex microsphere body with 0.5-5.0 μ m diameter of highly monodispersed diameter can obtain from following company: Polysciences, Inc., Warrington, PA 18976 and Interfaci-al Dynamics Corp., Portland, OR 97220.For these article of commerce, the diameter standard deviation (be the coefficient of deviation, or CV) that is expressed as the number percent of mean value is about 1% to 2%, and preferably CV surpasses 2%.
The CV of the light scattering pulse height distribution histogram that obtains from these spheroidal particles is the majorant of the focus dimension of the associated diameters of the sample flow of passing from FPA stream pipe and incident light (for example laser).If sample flow is relatively large, the particulate varying strength zone of incident beam of can flowing through produces a unnecessary big pulse height CV, even the CV of particulate itself is little so.Therefore will use the present invention best, it is basic keeping the narrow sample flow of constant size, below will tell about the method that reaches this purpose in detail.
Though optics capillary flow pipe central aperture diameter all is suitable for the present invention between 100 μ m-500 μ m, the central aperture diameter of 250 μ m is preferably.Preferably fluid sample stream is concentrated on the center, and with its diameter restrictions in the 3-10 mu m range.In an optimizer system, this diameter is about the 1%-3% of laser beam width.Under these conditions, can obtain CV value, and be best less than 2% light scattering pulse height distribution histogram about monodispersity poly microsphere.Used " monodispersity " is used to refer to the number of a class microsphere in the present context, and this class microsphere produces CV and is not more than 2% low-angle direct scattering optical pusle height distribution histogram.If do not control the sample flow diameter, so that the histogram CV according to monodisperse sphere shape particulate of non-immunochemistry sensitivity is about 2% or littler, then the sensitivity meeting of this method affects adversely, and is not easy to observe histogrammic symmetry and widens or constriction.
Can adopt the multiple fluid drive unit to obtain above-mentioned narrow liquid stream.Syringe pump and vacuum plant comprising Step-motor Control.Those skilled in the art will recognize that optimal mode is, wherein the liquid flow diameter can directly or indirectly be regulated in measuring process, and is controlled at the pattern in the suitable dimension scope.Also need to stream pipe be equipped with liquid stream centralization device in case as far as possible accurately constriction the fluid sample current limit at the center of sheath tubular type stream pipe.Monitor these two aspects of constriction and centralization, forward and backward during the immune response measuring fine particles or carry out in the monitoring sample flow all be possible.Can realize the either side of above two aspects with a class according to the monodispersity poly microsphere of number.The diameter of microsphere can be identical or different with microsphere with analysis, and do not participate in any immune association reaction.This class is contrasted the part that the property microsphere can be used as a kind of warning system, electronic sensing device in this system can sensing to the deviation of required CV value, feedback regulation liquid stream constriction device and liquid stream centralization device or one of them obtain required CV value with this then.
As mentioned above, for using the inventive method in conjunction with (sandwich construction) reaction among the embodiment at forward, bigger monodispersity microsphere and less polydispersity metal particle all will be with different complementary binding molecule bag quilts.At analyte is under antigen or the haptenic situation, monodispersity poly microsphere is by the antigen of first complementary type or haptens analyte quilt that antibody wraps, and this antibody can be monoclonal (monoclonal) antibody that points to antigen first epitope.The polydispersity metal particle is by the antibody sandwich of the second class antigen analyte, and this antibody can be monoclonal (monoclonal) antibody that points to the antigen analyte second class epitope.Under the situation that antigen exists, typical " sandwich construction " reaction takes place, antigen is linked to less metal particle on the surface of big poly microsphere randomly.When this situation takes place and the particle type immunocomplex that forms when passing aforementioned optical flow pipe thereupon, we are surprised to find that histogrammic CV and the standard deviation that is reflected by the histogram diagrammatic representation of single dispersed light scattering pulse height profile, and it has produced around peak-to-average and roughly has been the peak that symmetry has been widened.We find that also the concentration of analyte in this expansion and the fluid sample has direct and quantitative relation.
Fig. 1 has represented the principle of this embodiment of the present invention.What Figure 1A represented is the associated diameters of monodispersity latex microsphere body (the about 1.0 μ m of diameter) and polydispersity colloidal metal particulate (the about 20-120nm of diameter).Under the situation of above-mentioned preferable mean particle dia, the ratio of size that can see poly microsphere and metal particle is at 15-30: 1.This characteristic provides possibility for can be combined with a large amount of colloidal metal particulates randomly on each poly microsphere.Figure 1B represents the light scattering pulse height distribution histogram of latex microsphere body.The pulse height signal value height that sends from the latex microsphere body, and represent the narrower in width at histogrammic peak.This clearly illustrates that a little light scattering of sending from the metal particle from aggegation can not disturb mutually with the histogram that microsphere produces, and is promptly not overlapping.After metal particle was attached on the latex microsphere body under analyte intermediary, (Fig. 1 C) widened at the histogrammic peak of the latex in the forward association reaction.Even in conjunction with each embodiment of the present invention (histogram D1, D2 among Fig. 1 D, D3, D4) when analyzing the multiple analyte in the single sample simultaneously, the signal that unconjugated golden particulate produces does not have overlapping.
The general introduction that produces the method for stable absorption albumen in the latex microsphere surface can be with reference to Seaman, G.V.F., ed., Latex Based Technology in Diagnostics, Health ﹠amp; Science Communications, Washington, D.C.20005,1990.
We have found that the present invention also can carry out in an alternate embodiment.Realize in this way when of the present invention, respectively two types particulate and mutual combined molecule are hatched earlier and make this two based fine particles immunochemistry sensitization to form a kind of immune complex reaction thing that contains two kinds of compositions.In one embodiment, big poly microsphere is stably wrapped quilt by analyte.With the binding molecule bag of analyte complementation by metal particle.Two kinds of suspending liquid mix a kind of reagent of formation, can store earlier before use.Then this reagent is mixed with the fluid sample that comprises analyte.Utilize the formation of immunocomplex, the metal particle that analyte combines from poly microsphere displacement next part.This displacement causes that the dimension of the light scattering pulse height distribution histogram of above-mentioned immune complex reaction thing reduces.This reduce with fluid sample in the analyte concentration that exists proportional.
In a type of competition embodiment, analyte is being competed the metal particle that is attached to antibody one analyte bag quilt mutually with the poly microsphere of analyte bag quilt and is being got on.Through after suitable incubation period, the FPA of suspension and histogram just can have been measured.Only form according to thing by coated poly particulate.
As previously mentioned, in an inhibition type embodiment, analyte with hatched certain hour for example 5-30 minute by the polydispersity metal particle of anti-analyte binding molecule bag quilt, analyte is in conjunction with getting on and keeping apart a part of metal particle during this period.Unconjugated metal particle still can freely combine with the poly microsphere that adds subsequently and hatch, the former combined molecule of these poly microspheres (it can be a kind of carrier binding molecule that contains analyte) bag quilt, one period short time, after for example hatching in 5-10 minute, the FPA of the monodispersity poly microsphere of contrasting and testing just will show the amount of unconjugated metal particle, and this amount is inversely proportional to analyte concentration again conversely.
Obviously about the particulate of antigenicity or haptens analyte and antibody sandwich described easily forward of the present invention in conjunction with (sandwich construction) reaction, suppress, substitute and state of conflict embodiment, but should be understood that scope of the present invention has comprised that analyte wherein is the analysis that is present in the antibody in for example human serum and analog.Carrying out these with the forward combination when analyzing, two kinds of particulates can be used antigen coated with the analyte complementation, or randomly, a kind of particulate can be with antigen coated, another kind of second antibody bag quilt with aligning antibody analyte.In this mode, when having the antibody analyte to exist, little polydispersity metal particle and monodispersity poly particulate are compound, and the histogrammic aforementioned statistical of measuring light scattering pulse height profile changes.
With top the analysis of single analyte in the fluid sample has been described.Within the scope of the present invention, can analyze the multiple analyte in the same fluid sample simultaneously, and need must not separate sample to carry out the multiplicity analysis as the prior art method.This point can by distribute to diameter of each analyte to be determined and (or) refractive index and association reaction type be that unique poly microsphere is realized.Certainly, the bag of different big or small microspheres by situation by the analytic system type of distributing to analyte, for example forward in conjunction with, suppress, substitute or competition decides.The analytic system of multiple analyte can be identical or different.This embodiment of the present invention relies on the ministry of electronics industry of FPA to assign to monitor the light scattering signal that is produced by the different big or small poly microspheres of each class.It is on 883,574 the issued for approval U.S. Patent application that the electronic system of monitoring the microsphere of every kind of size is distributed in application numbers, can be for reference.In brief, two analytic systems or one of them can be analyzed the signal of sending from photodetector, and this photodetector receives the light scattering signal that poly microsphere sends.In an optimized analysis system based on software, the pulse of sending from light scattering detector enters a mould → number coverter, this converter is to the peak height sampling of each pulse, and these peak height values are sent into computing machine, computing machine is put these peak height values by size in order and they is lined up a histogram, it can be a level and smooth histogram, and each analyte has a figure.
The poly microsphere of binding molecule bag quilt can commercial buy that (Polysciences, Inc.), or by above Seaman, said method prepares in 1990.Be operated in the program at a typical bag, the buffer solution of microsphere suspension and certain density first binding molecule (PH is 7-8) is hatched a period of time, is typically 0.5-16 hour, is enough to make binding molecule to reach balance with combining of microsphere.By the microsphere of the coated mistake of simple centrifugation recovery, the solution that contains a kind of inert protein (for example degreaser drying milk particles or seralbumin) by the short time (for example 15 minutes) relatively exposes, and nonspecific binding site is blocked.Refrigeration damping fluid with at least 4 times of volumes washs coated microsphere 3 times at least then.Can adopt any storage damping fluid that can make the stable storage of microsphere suspension.Typical storage damping fluid is 0.5%BSA-0.1%NaN
3, in 0.154MNaCl, PH7.4 or 0.1%BSA-0.01NaN
3, in the 10mMHEPES damping fluid, PH7.5.
The polydispersity metallic colloid can be made by metal or metallic compound, for example metal oxide, metal hydroxides and slaine.Example comprises metallic gold, platinum, silver, copper, and is wherein golden optimum.The preparation method of the collaurum of required scope mean particle dia and can reference: Roth by the general introduction of the method for metal particle with the protein bag, J., " The Colloidal Gold Marker System for Light and Electron Microscopy Cytochemistry, " in Bullock, G.R.et al., Techniques in Immunochemistry, 2:217(19), in Horisberger, M., SEM11:9(1981), in Weiser, H.B., Inorganic Colloid Chemistry, J.Wiley, N.Y.1931, P.1, in Leuvering, J.H.W., United States Patent (USP) 4,313,734, with in Frens, G., Nature, Physical Science, 241:20(1973), more than all with for referencial use.
Polydispersity aurosol particulate suspension can be from E-YLaboratories, Inc., and San Matteo, CA94401 obtains, or by the described preparation of above-mentioned reference.Adopt above-mentioned Frens(1973 in a preferred method) method prepare the polydispersity aurosol particulate of mean particle dia size between 10-120nm.Though wider range of the golden particle size that this method produces, range size is not a key factor in the present invention.As mentioned above, the golden particulate of these sizes does not produce under condition of the present invention and can measure and interfering light scattering.Therefore, " polydispersity metal particle " as herein described refers to the number of the colloidal metal particulate of diameter between 20-120nm.
In a method, use K with antibody sandwich gold particulate
2CO
3Above-mentioned golden particulate solution is titrated to PH=7.5.The molecule that is used for wrapping quilt for example antibody is dissolved in the 10mMHEPES damping fluid of PH7.5, wherein contains 0.02%BSA and serves as stabilizing agent, and be added in the golden particulate solution by 1/10th volumes.Mix after 60 minutes, the 0.1% degreaser drying milk solid that adds 1/10th volumes is with the sealing nonspecific binding site.With centrifugal method particulate is being preserved damping fluid (10mM HEPES, PH7.5,0.1%BSA, 0.01%NaN
3, 1%mannitol(sweet mellow wine)) lining washing three times, exist side by side and promptly use or store down at 4 ℃.Bag is addressed in the reference of above Leuvering by other method of golden particulate.
Utilize the present invention, monoclonal by will be suitably complementary or polyclonal antibody is passive or covalently be attached to colloidal metal (as gold) particulate and poly (as latex) microsphere on, can carry out the high-sensitivity analysis many epitopes, high molecular antigen such as thyroid-stimulating hormone (TSH) in view of the above.If do not take into account the immunology globality of protein and under analysis condition, stably carry out combination, then can not strictly obtain the accurate method of protein bound.1.62 μ m diameter latex microsphere bodies (Interfacial Dynamics Corp.) with CV about 2% are especially tended in this analysis.Through the size of determination of electron microscopy at 50-80nm, have the aurosol particulate of polydispersity size to prepare by preceding method, be best.
The front said I have been found that when the colloidal metal particulate be when using under the high density at relative poly microsphere, as 2-100,000: 1, can serve as " cleanser ", be used for removing the non-specific interfering material that is prevalent in biogenic fluid sample (as serum) lining.That is to say that metal particle not only provides the basis of the quantitative immunoassay of the present invention, has reduced the adverse effect of interfering material simultaneously.Though see that from this respect effective particulate is wideer than very, the order of magnitude is 1000-10000: 1 metal particle is reasonable with the ratio of poly microsphere.
Following example only is used for illustrating several embodiments of the present invention, and desire does not limit scope of the present invention.Scope of the present invention is included in instructions and the wherein contained claim.
Example 1
The forward of serum TSH is in conjunction with (sandwich construction) response analysis
Adopt laxative remedy that polydispersity aurosol particulate (50-80nm) is wrapped quilt with the first anti-TSH monoclonal antibody (Kennebunkport, ME 04046 for BioDesign International, Inc.).With 0.2M sal tartari aurosol particulate suspension is transferred to PH7.5.Add the antibody that wraps quilt by 1/10th volumes in this suspension, it is at 10mMHEPES-0.02%BSA(PH=7.5) in diluted.Mix after 1 hour, mix the degreaser drying milk solid solution that adds 1/10th volumes.Utilize centrifugation that particulate is being contained 1%BSA, 0.01NaN
3, 1%mannitol(sweet mellow wine) 10mMHEPES damping fluid (PH7.5) in the washing three times.
Hansen, application number address in 883,574 the U.S. Patent application with the second anti-TSH monoclonal antibody bag by polystyrene (latex) pearl of 1.62 μ m diameters (diameter CV is about 2.0%), and be for reference at this.
By 10,000: 1 ratio hybrid packet by golden particulate and bag quilt present latex particulate, the formation reactant A.By certain volume reactant A is joined in the serum that contains TSH, make the final density of latex microsphere body be about 2 * 10
6/ mL, serum are 40%.After hatching 60 minutes under the room temperature, adopt the light scattering of low-angle forward in sheath tubular type FPA, to measure potpourri as described above.Fig. 3 has shown that standardization peak width unit (" NPW " among Fig. 2) is the function of analyte TSH concentration in the serum (concentration range is 0-1.2 μ IU/mL).The sensitivity of this analysis is about 5 * 10 in serum
-16M.
Example 2
The inhibition type analysis of thyroxine (T4)
By laxative remedy monodispersity latex microsphere body (diameter 1.62 μ m, Interfacial Dynamics Corp.) is wrapped quilt with human thyroglobulin (Calbiochem Inc.), form reactant A.Microsphere is at 10mM, in the HEPES damping fluid of PH7.5 with thyroglobulin antigen overnight incubation.Reclaim microsphere through centrifugation, with above-mentioned BSA and the NaN of containing
3HEPES damping fluid washing, be stored in the same damping fluid that contains sweet mellow wine.
By the antibody sandwich polydispersity aurosol particulate (50-80nm diameter) of laxative remedy, form reactant B with relative T4.In golden particulate, add the 10mMHEPES damping fluid (PH7.5) that contains antibody and BSA by 1/10th volumes.In T4 measured, what be used for wrapping quilt was a kind of polyclonal antibody (OEM Concepts) of T4 especially relatively through the IgG purifying.Coated particulate carries out back bag quilt with the solution of degreaser drying milk powder again, to seal the nonspecific binding site on it.Reclaim particulate through centrifugation, and with containing BSA and NaN
310mMHEPES damping fluid (PH7.5) washing.Particulate after the washing is stored in the same damping fluid, and adds sweet mellow wine as stabilizing agent.Coated golden particulate is diluted to a kind of analysis with in the damping fluid, and it contains 50mM glycocoll (PH9), 10mMEDTA, 0.01% naphthylamine sulfonic acid (Sigma Chem.Co., St.Louis, MO), 0.1%BSA, 0.3M KI and 0.01%NaN
3
The blood serum sample and the reactant B that contain T4 were hatched 30 minutes jointly, and adding reactant A then, to make its microsphere density be 2 * 10
7/ mL.After hatching 30 minutes again, come analytic sample in FPA with the histogram peak width of low-angle forward determination of light scattering sample.
T4 in the serum suppresses combining of golden particulate and colloidal state thyroglobulin.The serum T 4 of high concentration produces narrow histogram peak width, 4 of low concentration serum T opposite (Fig. 4 A).(San Luis Obispo CA) produces curve among Fig. 4 A, and is used as and standard EIA method for Biomerica, Inc., and promptly (Syva Corp., Palo Alto CA) carry out further comparative standard curve to the EMIT method by demarcating serum.Fig. 4 B has represented related data.Shown in correlativity in the analyte concentration scope greater than 95%.
Example 3
The simultaneity analysis of TSH in the same fluid sample and T4
Can analyze the multiple analysis thing for explanation in the same liquid sample, 2 li described TSH of example 1 and example and T4 immunoassay are combined.Be that the number of the monodispersity latex microsphere body of 0.95 μ m and 1.62 μ m is analyzed T4 and TSH with mean diameter respectively.The serum ultimate density is 15%, and the concentration of golden particulate is 1 * 10
10/ mL is 1 * 10 for T4 latex microsphere bulk concentration
8/ mL, and be 5 * 10 for TSH
6/ mL.
React in the following order.Use-case 1 described method is carried out the TSH analytical reactions.The reactant B that adds example 2 in this reaction after 30 minutes.After 25 minutes, add the reactant A of example 2 since then, and potpourri was hatched 5 minutes, carry out FPA then and analyze.
When analyzing the analyte of mid-strength range with FPA system and method for the present invention, be 4 μ g/mL to the result of T4, be 2 μ IU/mL to TSH.Do not have T4 and when containing the TSH sample of 2 μ IU/mL medium range levels, the T4 measured value is 0 with this method analysis, the TSH measured value is 2 μ IU/mL ± 10%.If use the TSH(0.3 μ IU/mL contain known low concentrations) and the sample of known intermediate concentration 4 μ g/mLT4, then the present invention and given value 10% with interior relevant.
Method of the present invention allows the simultaneity analysis.This description of test uses this method can obtain independently result, and in single process each do not have noticeable intercrossing to disturb between analyzing.
Example 4
The comparison of standard latex-latex agglutination method of the inventive method and mensuration IgE
For explanation the present invention effect, come analyst IgE with two kinds of forms in this one side of the adverse effect of eliminating non-specific binding.In first kind of form, will resist passive being coated on the present latex particulate of polyclonal antibody of IgE, carry out general latex microsphere body-latex microsphere body aggegation by people's such as aforementioned Masson method, and the disappearance situation of the single poly-microsphere of monitoring.The present latex particulate diameter is 1.62 μ m.Second kind of form is with forward association reaction embodiment of the present invention.
For wrapping, use 0.2MK by metal particle
2CO
3Golden particulate suspension is titrated to PH7.5.Add to be dissolved in this suspension by 1/10th volumes and contain 0.02%BSA, the anti-IgE(Biodesign lnternational of the mouse monoclonal people in the 10mMHEPES damping fluid of PH7.5, Inc., Kennebunkport, ME, 04046).Mix after 60 minutes, press the solution of the degreaser drying dry milk solids of 1/10th volumes adding 0.1%.Use the centrifugation separating particles,, be stored in and contain 10mMHEPES, 0.01%BSA, 0.01%NaN with same buffer washing three times
31%mannitol(sweet mellow wine) in the storage buffer.
For wrapping by the latex microsphere body, with 1.62 μ m latex microsphere body (Interfacial Dynamics Intnl., Portland, OR 97220) at 10mMHEPES, be diluted to 0.5% density in the PH7.5 solution, with this suspension and 100 μ g/mL affinity purifications goat anti-human IgE's solution 4 ℃ of following overnight incubation, isolate microsphere through centrifugation, and with HEPES damping fluid washing three times, be stored at last and contain 10mMHEPES, 0.1%BSA, 0.01%NaN
3, 1%mannitol(sweet mellow wine) damping fluid in.
For analyzing, latex and golden particulate are diluted to 1 * 10 respectively in analysis buffer
8/ mL and 2 * 10
10/ mL.The analyte blood serum sample aliquot of 50 μ l is added in the particle mixture of 450 μ l, uses vortex mixed.Final serum dilution is 10%.After hatching 15 minutes under 23 ℃, can carry out FPA to reaction mixture and analyze.Analysis buffer contains 0.05M glycocoll, 0.1%BSA, 0.3MKI, 0.01%NaN
3, PH9.5.
(Ventrex Laboratories, Portland ME) can be as the reference standard of above two kinds of forms for a kind of ELISA IgE analytical approach of SERUM IgE of commercialization.Fig. 5 A has represented the correlativity curve of latex-latex agglutination form, and Fig. 5 B then represents the correlation curve of the inventive method.Data can be clear that the correlativity of latex agglutination method is bad from figure, and sensitivity is limited, may be because the influence of interfering material in the analyte blood serum sample.With distinct contrast is that latex microsphere body of the present invention-Jin particulate method and reference method have outstanding correlativity (Fig. 5 B).
Example 5
The dynamics of histogram peak width expansion.
Carry out example 1 and analyze the low-angle forward light scattering FPA analysis that used method allows intermittent reaction mixture (containing 6 μ IU/mLTSH).
Under the situation that does not have analyte (-zero among Fig. 6-), the latex microsphere body that coated golden particulate is not joined to coated gets on, and the histogram dimension does not change yet.Yet, under the situation that analyte (among Fig. 6--) exists, when the histogram expansion of monodispersity latex microsphere body has reflection from first time point (2 minutes) in conjunction with detecting.This is widened in preceding 20 minutes of reaction and continues with the linear ratio, reaches plateau in the time of 100 minutes.
Claims (34)
1, a kind of poly particulate immune analysis method based on light scattering, be used at the antibody, antigen or the haptens analyte that do not have to measure simultaneously under the described poly particulate self aggregation situation one or more basically in an independent fluid sample, this method is made up of following steps:
A) be the monodispersity poly microsphere that each described analyte selection has the first binding molecule bag quilt of unique diameter or refractive index, like this can be from all other have and tell unique diameter microsphere histogram for the said light scattering pulse height distribution histogram of each class with microsphere of unique diameter or refractive index, this figure obtains from the optics sheath tubular type particle analysis gauge that flows;
B) equipped an adjustable apparatus that is used to produce narrow reaction mixture liquid stream on the mobile particle analysis gauge of optics sheath tubular type, one is used for and will flows through the adjustable apparatus of the described narrow adfluxion centre to centre heart of optics capillary sheath tubular type stream pipe, an incident light source, and the device of the said incident light scattering that causes by said microsphere of measurement, measure the single influence that disperse poly microsphere light scattering pulse height distribution histogram statistical change of each said analyte to the first binding molecule bag quilt in this optics sheath tubular type flows particle analysis gauge, described microsphere combines by the polydispersity colloidal metal particulate with the second binding molecule bag quilt and is initiated; With
C) the amount associated of each described analyte in the effect of described analyte and the single fluid sample is got up.
2, in accordance with the method for claim 1, wherein, said incident light scattering is the basic low-angle forward light scattering that is.
3, in accordance with the method for claim 1, wherein, said incident light scattering is to be the light scattering at right angle substantially.
4, in accordance with the method for claim 1, wherein, the polydispersion colloidal state metal particle that described analyte can increase the described second binding molecule bag quilt combines with the poly microsphere of the described first binding molecule bag quilt, and this increase causes the increase of the said dimension of described histogram.
5, by the said method of claim 1, the polydispersion colloidal state metal particle that wherein said analyte can reduce the described second binding molecule bag quilt combines with the poly microsphere of the described first binding molecule bag quilt, and this minimizing causes reducing of described histogrammic said dimension.
6, by the said method of claim 1, it is the figured standardization peak width of each described histogram that wherein said histogram dimension is formed.
7, by the said method of claim 1, comprise that further a step promptly provides a ratio of described metal particle and described poly microsphere to reduce the interference of non-specific binding material in described immunoassay effectively.
8, by the said method of claim 7, wherein said proportional range is 2-100,000: 1.
9, by the said method of claim 7, wherein said proportional range is 1,000-10,000: 1.
10, by the said method of claim 1, wherein said single poly microsphere that disperses is made up of homogeneous latex microsphere body.
11, by the said method of claim 1, wherein said microsphere is made up of about the microsphere of 0.02-100 mu m range mean diameter.
12, by the said method of claim 1, wherein said microsphere is made up of about the microsphere of 0.05-10.0 μ m left and right sides scope mean diameter.
13, by the said method of claim 1, wherein said microsphere is made up of the microsphere of mean diameter in the scope of the 0.5-5.0 μ m left and right sides.
14, by the said method of claim 1, wherein said colloidal metal particulate is selected from the group that comprises gold, platinum, silver, copper particulate.
15, by the described method of claim 1, wherein said colloidal metal particulate is golden particulate.
16, by the said method of claim 1, the mean diameter of wherein said polydispersion metal particle is about the 20nm-120nm scope.
17, by the described method of claim 1, the mean diameter of wherein said polydispersion metal particle is about the 50nm-80nm scope.
18, by the said method of claim 1, the diameter range of wherein said adjustable narrow sample flow is about 3 μ m-10 μ m, disperse one of poly microsphere generation to be not more than about 2% for the single of non-immunochemistry sensitization, around a figured deviation related coefficient of histogram mean value as reference.
19, by the described a kind of method of claim 1, constitute an analyte intermediary association reaction and comprise following each step:
A) list of putting into the described first binding molecule bag quilt in reaction vessel disperses the poly microsphere, the polydispersion colloidal state metal particle of the described second binding molecule bag quilt, with the said fluid sample that comprises described analyte, placing a period of time, is the resultant of reaction of monodispersity immunocomplex to form a kind of between three kinds of compositions effectively basically;
B) in described mobile particle analysis gauge, analyze described monodispersity immunocomplex;
C) the light scattering pulse height distribution histogram dimension of the described monodispersity immunocomplex of measurement;
D) c) the said dimension that obtains in the step and compare to measure c with reference to the dimension of reaction mixture) the step statistical that obtains described histogram dimension changes, this is made up of single poly microsphere that disperses of analyzing under the situation that does not have analyte or metal particle with reference to reaction mixture;
E) described analyte concentration associated in described histogram dimension statistical variation and the said fluid sample is got up.
20, by the described method of claim 19, wherein said analyte is antigen or haptens, and described first binding molecule is made up of the first complementary anti-analyte antibody, and said second binding molecule is made up of the second complementary anti-analyte antibody.
21, by the described method of claim 19, wherein said analyte is an antibody, described first and the antigen or the haptens of each a kind of naturally and said analyte complementary antibody of said second binding molecule, perhaps randomly, a kind of binding molecule is by forming with the antigen of described analyte complementary antibody, and another kind of binding molecule is made up of the second antibody of pointing to described analyte antibody.
22, by the described method of claim 1, the replacement reaction that constitutes a kind of analyte intermediary was made up of following each step:
A) will be mixed mutually with the polydispersion colloidal state metal particle of the binding molecule bag quilt of complementation by single poly microsphere that disperses of analyte bag quilt, perhaps randomly, mixing is by the poly microsphere of the binding molecule bag quilt of the metal particle of analyte bag quilt and complementation, to form a kind of monodispersity immune complex reaction thing;
B) described monodispersity immune complex reaction thing is mixed a period of time mutually with the fluid sample that contains described analyte, be enough to make described analyte to displace a part of said metal particle during this period of time from described monodispersity immunocomplex;
C) measuring described monodispersity immunocomplex adopts analyte to replace the light scattering pulse height distribution histogram dimension of front and back in step b); With
D) statistical of described histogram dimension is changed with described fluid sample in the analyte concentration associated get up.
23, by the described method of claim 22, wherein said analyte is antigen or haptens.
24, by the described method of claim 22, wherein said analyte is an antibody, and described complementary binding molecule is a corresponding antigen.
25, by the described method of claim 1, a kind of formation of state of conflict reaction is made up of following each step:
A) the polydispersity metal particle of the anti-analyte antibody sandwich of mixing, the monodispersity poly microsphere of analyte, antigen or haptens bag quilt, with contain analyte antigen or haptenic fluid sample, mix a period of time to be enough to form the competitive immunization complex;
B) measure the described coated single light scattering pulse height distribution histogram dimension of poly microsphere before and after described competitive immunization complex forms of disperseing; With
C) analyte concentration associated in described histogram dimension statistical variation and the fluid sample is got up.
26, by the described method of claim 1, a kind of formation of competitive reaction is made up of following steps:
A) the polydispersity metal particle of hybrid antigen or haptens bag quilt, the fluid sample that contains analyte antibody, with the monodispersity poly microsphere of quilt, mix a period of time to be enough to that the competitive immunization complex is formed with the binding molecule bag quilt of described antigen or haptens complementation;
B) measure the described coated single light scattering pulse height distribution histogram dimension of poly microsphere before and after described competitive immunization complex forms of disperseing; With
C) analyte concentration associated in described histogram dimension statistical variation and the fluid sample is got up.
27, by the described method of claim 1, a kind of formation that suppresses the type reaction is made up of following step:
A) disperse the poly microsphere with analyte or with the first binding molecule Sheet of analyte conjugation, form first kind of reactant;
B) use the second binding molecule bag with the analyte complementation by the polydispersity metal particle, form second kind of reactant;
C) hatch jointly with described second kind of reactant and the fluid sample that contains analyte, form a kind of polydispersity immunocomplex;
D) described first kind of reactant and described polydispersity immunocomplex are hatched jointly, to form a kind of monodispersity immune complex at described microsphere with between not in conjunction with that part of metal particle of analyte;
E) in described fluid fine particle analyser, analyze above-mentioned monodispersity immunocomplex;
F) dimension of the light scattering pulse height distribution histogram of the described monodispersity immunocomplex of measurement;
G) at f) the described dimension that obtains in the step and the corresponding comparison of reaction mixture of a reference, this reaction mixture be under no analyte or metal particle situation by bag by monodispersity poly microsphere forms, be determined at f with this) the statistical variation of the described histogram dimension that obtains in the step; With
H) statistical of described histogram dimension is changed with fluid sample in the analyte concentration associated get up.
28, by the described method of claim 27, wherein said analyte is haptens or antigen, and described second binding molecule is a kind of antihapten or anti-antigen-antibody.
29, by the described method of claim 27, wherein said analyte is a kind of antibody, described second binding molecule and described complementary antibody.
30, by the described method of claim 1, wherein in a single fluid sample, measure more than one analytes, have following each step to constitute:
A) specify a forward combination, replacement, inhibition or state of conflict reaction for each described analyte;
B) the polydispersity colloidal metal particulate of the second binding molecule bag quilt of the monodispersity poly microsphere of giving for first of the selected a kind of uniqueness of each said analyte to close molecule bag quilt and a kind of uniqueness;
C) in the same reaction container, carry out all described reactions;
D) measurement is changed by the statistical of each unique light scattering distribution of pulses histogram dimension of described each reaction generation; With
E) variation of above-mentioned each described histogram dimension and the concentration associated of each described analyte are got up.
31, by the described method of claim 19-30, wherein said histogram dimension is the standardization peak width in the described histogrammic diagrammatic representation.
32, in the case that separates, be used for measuring cover commercial detection articles for use of a kind of antibody, antigen or the haptens analyte of single fluid sample, it comprises:
A) mean diameter of binding molecule bag quilt is the monodispersity latex microsphere body of 0.5 μ m-10.0 μ m, and described latex microsphere body is no more than 2% around the related coefficient of the deviation of mean value; With
B) mean diameter of binding molecule bag quilt is the polydispersity aurosol particulate of 20-120nm; With optional,
C) reactant formed of the monodispersity immunocomplex that between coated monodispersity latex microsphere body and coated polydispersity gold particulate, forms.
33, in the mobile particle analysis gauge of the light scattering pulse height distribution histogram that contained monodispersity poly microsphere produces in can be detected and measure by fluid sample stream, sample flow flows through from the optics sheath tubular type of this instrument stream pipe, and the improvement of being done comprises to be provided described optics sheath tubular type stream pipe: one is used for will the flow through adjustable liquid driven side-gatherer of fluid sample stream of described stream pipe of constriction; One is used for the adjustable centralization device at the center that focuses on of the fluid sample stream of the constriction of stream flow tube; One is used for monitoring with reference to the single sensing device that disperses poly microsphere histogram dimension of property; With a feedback assembly that is used for controlling described liquid stream constriction device and described sample flow centralization device, remain in the required limit with this described reference histograms dimension.
34, it is in the diameter range to about the 3 μ m-10 μ m that the described analyser of claim 33, wherein said fluid sample flow described constriction, or is between about 1%-3% width of incident laser light beam.
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|---|---|---|---|
| US99490392A | 1992-12-22 | 1992-12-22 | |
| US994,903 | 1992-12-22 |
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| CN93119931A Expired - Fee Related CN1061142C (en) | 1992-12-22 | 1993-12-21 | Light scatter-based immunoassay without particle self aggregation |
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| US (1) | US5589401A (en) |
| EP (1) | EP0676045A4 (en) |
| JP (1) | JPH08505231A (en) |
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| AU (1) | AU670489B2 (en) |
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| US5079172A (en) * | 1988-11-04 | 1992-01-07 | Board Of Trustees Operating Michigan State University | Method for detecting the presence of antibodies using gold-labeled antibodies and test kit |
| CA2026409C (en) * | 1989-09-29 | 2004-11-09 | Ernest G. Schutt | Method of producing a reagent containing a narrow distribution of colloidal particles of a selected size and the use thereof |
| US5286452A (en) * | 1991-05-20 | 1994-02-15 | Sienna Biotech, Inc. | Simultaneous multiple assays |
-
1993
- 1993-06-15 TW TW082104758A patent/TW239881B/zh active
- 1993-12-21 CN CN93119931A patent/CN1061142C/en not_active Expired - Fee Related
- 1993-12-22 WO PCT/US1993/012428 patent/WO1994015193A1/en not_active Application Discontinuation
- 1993-12-22 JP JP6515382A patent/JPH08505231A/en active Pending
- 1993-12-22 KR KR1019950702661A patent/KR0171452B1/en not_active IP Right Cessation
- 1993-12-22 AU AU58734/94A patent/AU670489B2/en not_active Ceased
- 1993-12-22 EP EP94904875A patent/EP0676045A4/en not_active Withdrawn
- 1993-12-22 CA CA002151060A patent/CA2151060A1/en not_active Abandoned
-
1994
- 1994-08-05 US US08/286,778 patent/US5589401A/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110998294A (en) * | 2017-08-10 | 2020-04-10 | Jvc建伍株式会社 | Analysis method and analysis device |
| CN111398577A (en) * | 2020-06-08 | 2020-07-10 | 南京颐兰贝生物科技有限责任公司 | Quantitative immunoassay method |
Also Published As
| Publication number | Publication date |
|---|---|
| TW239881B (en) | 1995-02-01 |
| EP0676045A1 (en) | 1995-10-11 |
| EP0676045A4 (en) | 1998-02-04 |
| CA2151060A1 (en) | 1994-07-07 |
| US5589401A (en) | 1996-12-31 |
| KR0171452B1 (en) | 1999-05-01 |
| JPH08505231A (en) | 1996-06-04 |
| CN1061142C (en) | 2001-01-24 |
| AU670489B2 (en) | 1996-07-18 |
| AU5873494A (en) | 1994-07-19 |
| WO1994015193A1 (en) | 1994-07-07 |
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